Sructural study of immuno regulatory proteins by NMR spectroscopy

通过核磁共振波谱研究免疫调节蛋白的结构

• 批准号: 9357217
• 负责人: Ad Bax
• 金额: $ 22.57万
• 依托单位: NATIONAL INSTITUTE OF DIABETES AND DIGESTIVE AND KIDNEY DISEASES
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/9357217
• 关键词: Affinity; Antigen-Presenting Cells; Binding; Blood Circulation; Cell surface; Complex; Detection; Engineering; Family; Family member; Genes; Homologous Gene; Immune system; Immunoglobulins; In Vitro; J segment gene; Length; Lymphoid Tissue; Major Histocompatibility Complex; Maps; Mediating; Murid herpesvirus 1; NMR Spectroscopy; Nucleotides; Pathway interactions; Peptides; Peripheral; Process; Protein NMR Spectroscopy; Proteins; Recombinants; Relaxation; Signal Transduction; Site; Staging; T-Cell Development; T-Cell Receptor; T-Lymphocyte; Techniques; Thymus Gland; Vertebral column; adaptive immunity; base; genetic regulatory protein; insight; polypeptide; research study

As part of its strategy to evade detection by the host immune system, murine cytomegalovirus (MCMV) encodes three proteins that modulate cell surface expression of major histocompatibility complex class I (MHC-I) molecules: the MHC-I homolog m152/gp40, as well as the m02-m16 family members m04/gp34 and m06/gp48. Previous studies of the m04 protein revealed a divergent immunoglobulin (Ig)-like fold that is unique to immunoevasins of the m02-m16 family. Here, we engineer and characterize recombinant m06 and investigate its interactions with full length and truncated forms of the MHC-I molecule H2-Ld by several techniques. Furthermore, we employ solution NMR to map the interaction footprint of the m06 protein on MHC-I, taking advantage of a truncated H2-Ld, mini-H2-Ld, consisting of only the 12 platform domain. Mini-H2-Ld refolded in vitro with a high-affinity peptide to yields a molecule that shows outstanding NMR spectral features, permitting complete backbone assignments. These NMR-based studies reveal that m06 binds tightly to a discrete site located under the peptide-binding platform that partially overlaps with the 2-microglobulin (2m) interface on the MHC-I heavy chain, consistent with in vitro binding experiments showing significantly reduced complex formation between m06 and 2m-associated MHC-I. Moreover, we carry out NMR relaxation experiments to characterize the ps-ns dynamics of the free mini-H2-Ld MHC-I molecule, revealing that the site of interaction is highly ordered. This study provides insight into the mechanism of the interaction of m06 with MHC-I, suggesting a structural manipulation of the target MHC-I molecule at an early stage of the peptide-loading pathway.

作为逃避宿主免疫系统检测的策略的一部分，小鼠巨细胞病毒（MCMV）编码三种调节细胞表面主要组织相容性复合体I类（MHC-I）分子表达的蛋白：MHC-I同源物m152/gp40，以及m02-m16家族成员m04/gp34和m06/gp48。先前对m04蛋白的研究揭示了m02-m16家族免疫球蛋白独有的发散性免疫球蛋白（Ig）样折叠。在这里，我们设计和表征重组m06，并通过几种技术研究其与MHC-I分子H2-Ld的全长和截短形式的相互作用。此外，我们利用截断的H2-Ld， mini-H2-Ld，仅由12个平台结构域组成，利用溶液核磁共振绘制了m06蛋白在MHC-I上的相互作用足迹。Mini-H2-Ld与高亲和肽在体外重新折叠，产生的分子显示出出色的核磁共振光谱特征，允许完整的骨架分配。这些基于核磁共振的研究表明，m06与位于肽结合平台下的一个离散位点紧密结合，该位点与MHC-I重链上的2-微球蛋白（2m）界面部分重叠，这与体外结合实验一致，表明m06与2m相关的MHC-I之间的复合物形成显著减少。此外，我们进行了核磁共振弛豫实验来表征自由的mini-H2-Ld MHC-I分子的ps-ns动力学，揭示了相互作用的位置是高度有序的。这项研究揭示了m06与MHC-I相互作用的机制，表明在多肽装载途径的早期阶段，靶MHC-I分子的结构操纵。